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QIAamp DNA FFPE Advanced Kits

Next-generation DNA isolation for formalin-fixed paraffin-embedded (FFPE) tissues

Features

  • High recovery of amplifiable DNA
  • Paraffin removal without xylene or similar solvents
  • Uracil (deaminated cytosine)–artifact removal step using uracil-N-glycosylase (UNG) during DNA extraction
  • Ready-to-use DNA for PCR, digital PCR (dPCR) and next-generation sequencing (NGS)

Products

QIAamp DNA FFPE Advanced Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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QIAamp DNA FFPE Advanced Kit (50)

Cat. No. / ID: 56604

For 50 preps: QIAamp UCP MinElute Columns, collection tubes, Deparaffinization Solution, Proteinase K, RNase A, RNase-free water and buffers
CHF 441.00
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QIAamp DNA FFPE Advanced UNG Kit (50)

Cat. No. / ID: 56704

For 50 preps: Uracil-N-glycosylase, QIAamp UCP MinElute columns, collection tubes, Deparaffinization Solution, Proteinase K, RNase A, RNase-free water and buffers
CHF 515.00
Add to cart
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Uracil-N-Glycosylase (2 x 1 ml)

Cat. No. / ID: 19160

50 preps: For use with QIAGEN DNA FFPE UNG kits
CHF 197.00
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Product Details

Increase your recovery of high-quality DNA from FFPE tissue with the QIAamp DNA FFPE Advanced Kits’ xylene-free, no-wash deparaffinization, double-lyse protocol, and UCP (ultra-clean production) spin column technology.

Remove C→U transitions of nucleic acids with the kits’ optional UNG digest to optimize your recovered DNA for NGS analysis.

You can also automate the QIAamp DNA FFPE Advanced protocols on the QIAcube Connect.

Performance

One characteristic of FFPE DNA quantification is that different methods will give out different results, and high UV-vis or fluorometric values do not necessarily mean good PCR performance.

Because real-time PCR, or quantitative PCR (qPCR), is one of the most common downstream applications for FFPE, the QIAamp DNA FFPE Advanced Kits are optimized for qPCR.

Regardless of values obtained in UV-vis or fluorometric measurements, QIAamp DNA FFPE Advanced Kits consistently showed better PCR performance under qPCR quantification than the other products tested (see figure “ Optimized for PCR performance”).

The QIAamp DNA FFPE Advanced UNG Kit is also well suited for purifying DNA to be used in NGS analysis, because it addresses the issue of artificial C→T/G→A transitions, which commonly occur in FFPE material due to cytosine deamination (see “ Artificial C→T/G→A transitions”).

Through UNG-based uracil digestion during DNA isolation, the QIAamp DNA FFPE Advanced UNG protocol reduces false-positive reports of single-nucleotide variants (SNVs) in NGS (see figure “ Dramatic reduction in artifactual C→T | G→A transitions”).

See figures

Principle

There are three major challenges in preparing DNA from FFPE tissues:

  • Low yields due to limited input material and compromised DNA
  • Nonamplifiable DNA due to formalin-induced crosslinks
  • Deaminated-cytosine artifacts can cause false results in NGS for mutational analyses

The QIAamp DNA FFPE Advanced Kits maximize DNA yields from limited sample inputs in two ways:

  • By implementing a two-step lysis procedure, which ensures high DNA extraction even from difficult-to-lyse samples
  • By replacing solvent-based paraffin removal with the use of Deparaffinization Solution, which eliminates all wash steps before initial lysis, thus minimizing the risk of losing scarce sample material

Crosslink removal further increases the recovery of amplifiable DNA (see figure “ Optimized for PCR performance”).

Optional UNG treatment before the second lysis removes deaminated-cytosine artifacts, making the DNA especially suitable for NGS analysis (see table “ Primed for NGS”, as well as figures “ Reliable dPCR and NGS results” and “ Dramatic reduction in artifactual C→T | G→A transitions”).

See figures

Procedure

The QIAamp DNA FFPE Advanced procedure consists of a simplified deparaffinization step, two lysis steps with in-between de-crosslinking and optional artifact removal, and the standard bind-wash-elute steps (see figure “ QIAamp DNA FFPE Advanced workflow”).

See figures

Applications

DNA from FFPE using the QIAamp DNA FFPE Advanced Kits can be used immediately for PCR, dPCR or NGS, or it can be stored at −30 to −15°C.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsPCR, digital PCR, next-generation sequencing
Elution volume20–100 µl
FormatSpin column
Main sample typeFormalin-fixed paraffin-embedded (FFPE) tissue samples
ProcessingManual or automated with the QIAcube Connect
Purification of total RNA, miRNA, poly A+ mRNA, DNA or proteinGenomic DNA
Sample amountTissue sections, each with a thickness of 5–10 µm, for a total volume of 4&nbsp;mm<sup>3</sup>
TechnologySilica technology

Resources

Brochures & Guides (3)
Sample to Insight solutions for successful molecular analysis
Critical factors for molecular analysis of FFPE samples
Kit Handbooks (1)
Application/Protocol Documents (2)
Safety Data Sheets (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
Quick-Start Protocols (1)

FAQ

Is the DNA extracted with the QIAamp DNA FFPE Advanced Kits suitable for downstream applications that require more intact DNA such as long range PCR (>1 kb) and long read DNA sequencing?
DNA from FFPE samples is often fragmented, yielding DNA with a broad size distribution depending on multiple factors such as fixation and storage conditions.

The QIAamp DNA FFPE Advanced Kits provide an optimized workflow for extraction of DNA for use in short amplicon PCR, dPCR and next-generation sequencing analysis using targeted DNA panels. 
In case FFFPE samples are of high quality and allow the extraction of more intact DNA we offer a supplementary protocol for downstream applications that require larger DNA fragments. These applications include for instance large amplicon PCR (>0,5kb) and long read DNA sequencing.
High DNA integrity can be presumed if formalin fixation was less than 24 hours and the sample has been stored at low temperature (4-8°C or - 20°C) or for a short period of time (e.g. up to a few weeks). For these samples the  Supplementary Protocol “extraction of more intact DNA from FFPE tissue material using the QIAamp DNA FFPE Advanced Kits with Buffer LF” is suitable as it protects and best preserves the DNA size distribution present in the original FFPE sample during DNA extraction.
Please contact TechService to inquire about the supplementary protocol and the availability of Buffer LF.
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Can the second Proteinase K lysis step in the QIAamp DNA FFPE Advanced procedure be carried out at lower temperatures than 65°C?
A temperature range of 56°C - 68°C works fine for the second Proteinase K lysis step. However, we recommend following the instructions in the QIAamp DNA FFPE Advanced Handbooks and perform the second lysis step at 65°C.
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What is the difference of buffer FTB versus Buffer ATL?
Buffer FTB provides optimized lysis conditions, and additionally allows the specific removal of deaminated cytosine residues by the enzyme Uracil-N-Glycosylase (UNG) in the QIAamp DNA FFPE Advanced UNG workflow.
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Can the 2nd Prot K step be omitted?
The 2nd Proteinase K step improves lysis efficiency and yields, in particular for difficult-to-lyse tissue material. Hence, omission of this step may result in decreased yields.
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Are there important considerations for plasma generation and urine handling?

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699
What size of DNA can be expected?

Size distribution of the extracted DNA can vary significantly and first and foremost strongly depends on the DNA quality present in the original FFPE sample. Formalin-fixation, paraffin-embedding and storage conditions are factors that affect the DNA size distribution and may cause significant fragmentation of nucleic acids. To limit the extent of nucleic acid fragmentation, be sure to:

  • Fix tissue samples in 4%–10% formalin as quickly as possible after surgical removal.
  • Use a fixation time of 14–24 h (longer fixation times lead to more severe DNA fragmentation, resulting in poor performance in downstream assays).
  • Thoroughly dehydrate samples prior to embedding (residual formalin can inhibit proteinase K digestion)
  • Store FFPE tissue samples at 4-8°C
153897
What are recommended stopping points in the procedure of the QIAamp DNA FFPE Advanced Kits?
After 90°C incubation for cross-link removal sample lysates can be stored at 4-8°C for up to one week and at -20°C or -80°c for up to 4 weeks. The upper blue phase of Deparaffinization Solution should be removed before storage. Before proceeding with the workflow after storage thaw frozen samples at room temperature for 30 minutes or allow refrigerated (4-8°C) samples to equilibrate to room temperature for 15-30 minutes.
153892
Can samples be lysed overnight with Proteinase K in the QIAamp DNA FFPE Advanced procedure?
If it is more convenient either the first or the second Proteinase K lysis step in the QIAamp DNA FFPE Advanced workflow can be performed overnight. This will not affect the DNA quality but also not increase yields.
153893